Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. Coxiella's former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, our seminal advance of host cell-free (axenic) growth of Coxiella in acidified citrate cysteine medium (ACCM) enabled us to quickly develop a a complete genetics tool box. Most recently, we developed a defined medium that supports robust growth of Coxiella called ACCM-D that contains amino acids as sole carbon and energy sources. Coxiella is auxotrophic for Arg and Lys by lacking the final steps in biosynthesis. Heterologous expression of Legionella pneumophila argGH and lysA rescues growth in Arg and Lys dropout media, respectively, thus providing two methods for nutritional selection of Coxiella transformants. This allows strong, non-antibiotic-based selection of genetic transformants, an important advance considering selectable markers based on antibiotic resistance are limited for this select agent. Collectively, our repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Indeed, we have constructed knockout strains in both virulent and avirulent Coxiella, including those with deletions in genes encoding components of the Dot/Icm type IVB secretion system (T4BSS) and secreted proteins. These studies have confirmed that T4BSS function is critical for Coxiella growth in macrophages. Moreover, using Cre-lox, we have created a 32.4 kb dot/icm mutant of the virulent Nine Mile phase I strain that lacks the entire dot/icm locus required for synthesis of the T4BSS. The mutant displays vigorous growth in synthetic medium but cannot grow intracellularly. The strain expresses phase I LPS necessary for vaccine efficacy, which will be tested in a Guinea pig model. Mutational analysis has also identified genetic mechanisms of LPS phase variation associated with virulence. Coxiella encodes a paucity of transcriptional regulators that are likely critical for intramacrophage survival and/or developmental transitions. The PhoBR two-component system of Coxiella is especially intriguing as homologous systems in other bacteria regulate virulence gene expression. Moreover, the role the stationary phase sigma factor RpoS in stress survival is unknown. Unraveling PhoBR and RpoS regulatory networks will identify important virulence determinants. Using gene knockouts, reporter assays, RNAseg, and whole bacterial proteome mass spectrometry, we resolved regulatory cascades of RpoS and PhoBR. The only genetic lesions proven to result in attenuated Coxiella virulence in an immunocompetent animal model are associated with defective LPS synthesis. Virulent phase I organisms with full-length LPS transition to avirulent phase II organisms with severely truncated LPS upon repeated in vitro passage. Given the critical role of LPS in Coxiella virulence, it is important to understand the molecular basis of phase variation. We used allelic exchange and complementation to genetically define pathways of LPS phase conversion. Coxiella undergoes an intracellular biphasic developmental cycle that generates two distinct morphological variants that can be distinguished by ultrastructure and protein composition. Small cell variants (SCV) do not replicate, contain condensed chromatin, and are considered extracellular survival forms. SCV differentiate into replicative large cell variants (LCV) with dispersed chromatin. Transition of LCV back to SCV occurs coincident with Coxiella entry into stationary growth phase, with nearly homogeneous SCV present upon extended incubation (2 to 4 weeks) of infected cell cultures. As an amenable model to help better understand the biological relevance of Coxiella differentiation, we established that SCV/LCV transitions are recapitulated by organisms growing in the third-generation axenic media, ACCM-D. This discovery enables studies of Coxiella developmental biology without experimental difficulties encountered with host cell-propagated bacteria. Comparative transcriptomics and proteomics of LCV and SCV have now revealed molecular determinants of morphological differentiation that likely contribute to the unique biological characteristics of cell forms. Genes associated with differentiation are now being inactivated and mutants phenotyped. We found that Coxiella genetic diversity correlates with disease pathogenesis. Thirteen strains within the six major genetic groups of Coxiella were evaluated for pathogenicity in a guinea pig model of infection. Based on temperature and splenomegaly, strains grouped as high, intermediate, or low/no virulence. Two extremes in pathogenesis are the highly virulent NMI tick isolate (Group 1) and the avirulent Dugway rodent isolate (group VI). Interestingly, animals infected with Dugway still seroconvert, indicating productive infection. To gain insight into pathotype-specific virulence, we generated mutants for virulence testing.